Nitrogenous heterocylic compounds

ABSTRACT

The present invention relates to nitrogen-containing heterocyclic compounds and pharmaceutically acceptable salts thereof which have inhibitory activity on the phosphorylation of kinases, which inhibits the activity of such kinases. The invention is also related to a method of inhibiting kinases and treating disease states in a mammal by inhibiting the phosphorylation of kinases. In a particular aspect the present invention provides nitrogen-containing heterocyclic compounds and pharmaceutically acceptable salts thereof which inhibit phosphorylation of a PDGF receptor to hinder abnormal cell growth and cell wandering, and a method for preventing or treating cell-proliferative diseases such as arteriosclerosis, vascular reobstruction, cancer and glomerulosclerosis.

TECHNICAL FIELD

[0001] The present invention relates to nitrogen-containing heterocycliccompounds and pharmaceutically acceptable salts thereof which haveinhibitory activity on the phosphorylation of kinases, which inhibitsthe activity of such kinases. The invention is also related to a methodof inhibiting kinases and treating disease states in a mammal byinhibiting the phosphorylation of kinases.

BACKGROUND ART

[0002] PDGF (platelet-derived growth factor) is known to act as anaggravating factor for cell-proliferative diseases such asarteriosclerosis, vascular reobstruction after percutaneous coronaryangioplasty and bypass operation, cancer, glomerulonephritis,glomerulosclerosis, psoriasis and articular rheumatism [Cell, 46,155-169 (1986); Science, 253, 1129-1132 (1991); Nippon Rinsho (JapaneseJ. of Clinical Medicine), 50, 3038-3045 (1992); Nephrol Dial Transplant,10, 787-795 (1995); Kidney International, 43 (Suppl. 39), 86-89 (1993);Journal of Rheumatology, 21, 1507-1511 (1994); Scandinavian Journal ofImmunology, 27, 285-294 (1988), etc.].

[0003] As for quinazoline derivatives which are useful as drugs,N,N-dimethyl-4-(6,7-dimethoxy-4-quinazolinyl)-1-piperazine carboxamideis described as a bronchodilator in South African Patent No. 67 06512(1968). Dimethoxyquinazoline derivatives are described as inhibitors ofphosphorylation of epidermal growth factor (EGF) receptor in JapanesePublished Unexamined Patent Application No. 208911/93 and WO 96/09294.Quinoline derivatives having benzodiazepin receptor agonist activity aredescribed in Pharmacology Biochemistry and Behavior, 53, 87-97 (1996)and European Journal of Medicinal Chemistry, 31, 417-425 (1996), andquinoline derivatives which are useful as anti-parasite agents aredescribed in Indian Journal of Chemistry, 26B, 550-555 (1987).

[0004] Inhibitors of phosphorylation of PDGF receptor so far knowninclude bismono- and bicyclic aryl compounds and heteroaryl compounds(WO 92/20642), quinoxaline derivatives [Cancer Research, 54, 6106(1994)], pyriinidine derivatives (Japanese Published Unexamined PatentApplication No. 87834/94) and dimethoxyquinoline derivatives [Abstractsof the 16th Annual Meeting of the Pharmaceutical Society of Japan(Kanazawa) (1996), 2, p. 275, 29(C2) 15-2].

DISCLOSURE OF THE INVENTION

[0005] An object of the present invention is to providenitrogen-containing heterocyclic compounds and pharmaceuticallyacceptable salts thereof which have inhibitory activity on thephosphorylation of kinases, which inhibits the activity of the kinases.Particularly, important kinase inhibition according to the invention isof receptor tyrosine kinases including platelet-derived growth factor(PDGF) receptor, Flt3, CSF-1R, epidermnal growth factor receptor (EGRF),fibroblast growth factor (FGF), vascular endothelial growth factorreceptor (VEGFR) and others. Another class of kinase inhibitionaccording to the invention is inhibitory activity nonreceptor tyrosinekinases including src and abl, and the like. A third class of kinaseinhibition according to the invention is inhibitory activity towardserine/threonine kinases, including such kinases as MAPK, MEK and cyclindependent kinases (CDKs) that mediate cell prolifetation, AKT and CDKsuch that mediate cell survival and NIK that regulate inflammatoryresponses. Inhibition of such kinases can be used to treat diseasesinvolving cell survival, proliferation and rnigration, includingcardiovascular disease, such as arteriosclerosis and vascularreobstruction, cancer, glomerulosclerosis fibrotic diseases andinflammation, as well as the general treatment of cell-proliferativediseases.

[0006] In a preferred embodiment, the present invention providescompounds and pharmaceutically acceptable salts thereof which inhibit orprevent inhibition of phosphorylation of at least one PDGF receptor byat least one tyrosine kinase. Such PDGF receptor kinase inhibition canhinder abnormal cell growth and cell wandering, and thus such compoundsare useful for the prevention or treatment of cell-proliferativediseases such as arteriosclerosis, vascular reobstruction, cancer andglomeruloscierosis.

[0007] The present invention relates to nitrogen-containing heterocycliccompounds represented by formula I as follows:

[0008] wherein

[0009] R¹ is a member selected from the group consisting of:

[0010] —CN,—O—C₁₋₈ alkyl that is a straight or branched chain,—O-phenyl, —O-naphthyl,

[0011] —O-indolyl and —O-isoquinolinyl;

[0012] R² and R⁴ are each independently a member selected from the groupconsisting of:

[0013] hydrogen, —O(—CH₂)—CH₃, —O—CH₂—CH═CH₂, —O—CH₂—C4CH and—O(—CH₂)_(n)—R³;

[0014] providing that one of the R² and R⁴ groups is hydrogen and theremaining R² or R⁴ group is other than hydrogen;

[0015] n is 1, 2or3;

[0016] R³ is a member selected from the group consisting of:

[0017] —OH, —O—CH₃, —O—CH₂—CH₃, —NH₂, —N(—CH₃)₂,—NH(—CH₂-phenyl),—NH(-Phenyl), —CN

[0018] and all pharmaceutically acceptable isomers, salts, hydrates,solvates and prodrug derivatives thereof

[0019] Particularly preferred compounds according to formula above aresuch compounds wherein R¹ is a member selected from the group consistingof CN, —O-methyl, —O-ethyl, —O-propyl, —O-isopropyl, —O-butyl,—O-t-butyl, —O-isoamyl, 1-naphthyloxy, 2-naphthyloxy, 4-indolyloxy,5-indolyloxy, 5-isoquinolyloxy, and position isomers and homologsthereof, and all pharmaceutically acceptable isomers, salts, hydrates,solvates and prodnig derivatives of such compounds.

[0020] The pharmaceutically acceptable salts of the compounds accordingto formula (I) include pharmaceutically acceptable acid addition salts,metal salts, ammonium salts, organic amine addition salts, amino acidaddition salts, etc. Examples of the pharmaceutically acceptable acidaddition salts of the compounds of formula (I) are inorganic acidaddition salts such as hydrochloride, sulfate and phosphate, and organicacid addition salts such as acetate, maleate, fumarate, tartrate,citrate and methanesulfonate. Examples of the pharmaceuticallyacceptable metal salts are alkali metal salts such as sodium salt andpotassium salt, alkaline earth metal salts such as magnesium salt andcalcium salt, aluminum salt and zinc salt. Examples of thepharmaceutically acceptable ammonium salts are ammonium salt andtetramethyl ammonium salt. Examples of the pharmaceutically acceptableorganic amine addition salts include heterocyclic amine salts such asmorpholine and piperidine salts. Examples of the pharmaceuticallyacceptable amino acid addition salts are salts with lysine, glycine andphenylalanine.

[0021] In a preferred embodiment the invention provides compoundsaccording to formula I(a) and formula I(b) as follows:

[0022] wherein

[0023] R¹ is a member selected from the group consisting of:

[0024] —CN, —O—C₁₋₈ alkyl that is a straight or branched chain,—O-phenyl, —O-naphthyl, —O-indolyl and —O-isoquinolinyl;

[0025] and all pharmaceutically acceptable isomers, salts, hydrates,solvates and prodrug derivatives thereof.

[0026] In another preferred embodiment the invention provides compoundsaccording to formula (Ic) and formula (Id) as follows:

[0027] wherein

[0028] R¹ is a member selected from the group consisting of:

[0029] —CN, —O—C₁₋₈ alkyl that is a straight or branched chain,—O-phenyl, —O-naphthyl, —O-indolyl and —O-isoquinolinyl;

[0030] and all pharmaceutically acceptable isomers, salts, hydrates,solvates and prodrug derivatives thereof.

[0031] In still another preferred embodiment the invention providescompounds according to formula I(e) and formula I(f) as follows:

[0032] wherein

[0033] R¹ is a member selected from the group consisting of:

[0034] —CN, —O—C₁₋₈ alkyl that is a straight or branched chain,—O-phenyl, —O-naphthyl, —O-indolyl and —O-isoquinolinyl;

[0035] and all pharmaceutically acceptable isomers, salts, hydrates,solvates and prodrug derivatives thereof.

[0036] In yet another preferred embodiment the invention providescompounds according to formula I(g) and formula I(h) as follows:

[0037] wherein

[0038] R¹ is a member selected from the group consisting of:

[0039] —CN, —O—C₁₋₈ alkyl that is a straight or branched chain,—O-phenyl, —O-naphthyl, —O-indolyl and —O-isoquinolinyl;

[0040] R³ is a member selected from the group consisting of:

[0041] —OH, —O—CH₃, —O—CH₂—CH₃, —NH₂, —N(—CH₃)₂,—NH(—CH₂-phenyl),—NH(-Phenyl), —CN

[0042] and all pharmaceutically acceptable isomers, salts, hydrates,solvates and prodrug derivatives thereof.

[0043] The pharmaceutically acceptable salts of the compounds accordingto formula (I) include pharmaceutically acceptable acid addition salts,metal salts, ammonium salts, organic amine addition salts, amino acidaddition salts, etc.

[0044] The present invention is not limited by the above listedcompounds. Analogs of the bicyclic compounds are contemplated.

[0045] Further, an especially preferred embodiment of the presentinvention is a compound selected from the group consisting of:

[0046]N-[4-(methylethoxy)phenyl]{4-[7-(2-morpholin-4-ylethoxy)quinazolin-4-yl]piperazinyl}carboxamide

[0047]N-(4-cyanophenyl){4-[7-(2-morpholin-4-ylethoxy)quinazolin-4-yl]piperazinyl}carboxamide

[0048]N-(4-cyanophenyl){4-[7-(2-pyrrolidinylethoxy)quinazolin-4-yl]piperazinyl}carboxamide

[0049]N-[4-(methylethoxy)phenyl]{4-[7-(2)pyrrolidinylethoxy)quinazolin-4-yl]piperazinyl}carboxamide

[0050]N-[4-(methylethoxy)phenyl]{4-[7-(2-piperidylethoxy)quinazolin-4-yl]piperazinyl}carboxamide

[0051]{4-[7-(2-methoxyethoxy)quinazolin-4-yl]piperazinyl}-N-[4-(methylethoxy)phenyl]carboxamide

[0052]N-(4-cyanophenyl){4-[7-(2-methoxyethoxy)quinazolin-4-yl]piperazinyl}carboxamide

[0053]N-(4-cyanophenyl){4-[7-(2-piperidylethoxy)quinazoin-4-yl]piperazinyl}carboxamide

[0054]{4-[7-(2-methoxyethoxy)quinazolin-4-yl]piperazinyl}-N-(4-phenoxyphenyl)carboxamide

[0055]N-(4-indol-5-yloxyphenyl){4-[7-(2-methoxyethoxy)quinazolin-4-yl]piperazinyl}carboxamide

[0056]N-(4-(5-isoquinolyloxy)phenyl){4-[7-(2-methoxyethoxy)quinazolin-4-yl]piperazinyl}carboxamide

[0057]N-[4-(methylethoxy)phenyl]{4-[7-(3-piperidylpropoxy)quinazolin-4-yl]piperazinyl}carboxamide

[0058]{4-[7-(2-methoxypropoxy)quinazolin-4-yl]piperazinyl}-N-(4-phenoxyphenyl)carboxamide

[0059]N-(4-indol-5-yloxyphenyl){4-[7-(2-methoxypropoxy)quinazolin-4-yl]piperazinyl}carboxamide

[0060]N-(4-(5-isoquinolyloxy)phenyl){4-[7-(2-methoxypropoxy)quinazolin-4-yl]piperazinyl}carboxamide

[0061]N-(4-cyanophenyl){4-[7-(3-piperidylpropoxy)quinazolin-4-yl]piperazinyl}carboxamide

[0062]N-[4-(methylethoxy)phenyl]{4-[7-(3-morpholin-4-ylpropoxy)quinazolin-4-yl]piperazinyl}carboxamide

[0063]N-(4-cyanophenyl){4-[7-(3-morpholin-4-ylpropoxy)quinazolin-4-yl]piperazinyl}carboxamide

[0064]N-[4-(methylethoxy)phenyl]{4-[7-(3-pyrrolidinylpropoxy)quinazolin-4-yl]piperazinyl}carboxamide

[0065]N-(4-cyanophenyl){4-[7-(3-pyrrolidinylpropoxy)quinazolin-4-yl]piperazinyl}carboxamide

[0066]N-(4-cyanophenyl){4-[7-(3-methoxypropoxy)quinazolin-4-yl]piperazinyl}carboxamide

[0067]{4-[7-(3-methoxypropoxy)quinazolin-4-yl]piperazinyl}-N-[4-(methylethoxy)phenyl]carboxamide

[0068] and all pharmaceutically acceptable isomers, salts, hydrates,solvates and prodrug derivatives thereof.

[0069] The compounds may be prepared using methods and procedures asgenerally described in WO 98/14431 published Sep. 12, 1998, which isincorporated herein by reference. Starting materials may be made orobtained as described therein as well.

[0070] Leaving groups such as halogen, lower alkoxy, lower alkylthio,lower alkylsulfonyloxy, arylsulfonyloxy, etc, may be utilized whennecessary except for the reaction point, followed by deprotection.Suitable amino protective groups are, for example, those described in T.W. Greene, Protective Groups in Organic Synthesis, John Wiley & SonsInc. (1981), etc., such as ethoxycarbonyl, t-butoxycarbonyl, acetyl andbenzyl. The protective groups can be introduced and eliminated accordingto conventional methods used in organic synthetic chemistry [e.g., T. W.Greene, Protective Groups in Organic Synthesis, John Wiley & Sons Inc.(1981)].

[0071] In such processes, if the defined groups change under theconditions of the working method or are not appropriate for carrying outthe method, the desired compound can be obtained by using the methodsfor introducing and eliminating protective groups which areconventionally used in organic synthetic chemistry [e.g., T. W. Greene,Protective Groups in Organic Synthesis, John Wiley & Sons Inc. (1981)],etc. Conversion of functional groups contained in the substituents canbe carried out by known methods [e.g., R. C. Larock, ComprehensiveOrganic Transformations (1989)] in addition to the above-describedprocesses, and some of the active compounds of formula I may be utilizedas intermediates for further synthesizing novel derivatives according toformula I.

[0072] The intermediates and the desired compounds in the processesdescribed above can be isolated and purified by purification methodsconventionally used in organic synthetic chemistry, for example,neutralization, filtration, extraction, washing, drying, concentration,recrystallization, and various kinds of chromatography. Theintermediates may be subjected to the subsequent reaction withoutpurification.

[0073] There may be tautomers for some formula I, and the presentinvention covers all possible isomers including tautomers and mixturesthereof. Where chiral carbons lend themselves to two differentenantiomers, both enantiomers are contemplated as well as procedures forseparating the two enantiomers.

[0074] In the case where a salt of a compound of formula I is desiredand the compound is produced in the form of the desired salt, it can besubjected to purification as such. In the case where a compound offormula I is produced in the free state and its salt is desired, thecompound of formula I is dissolved or suspended in a suitable organicsolvent, followed by addition of an acid or a base to form a salt.

[0075] The following non-limiting reaction Schemes I, II and IIIillustrate preferred embodiments of the invention with respect to makingcompounds according to the invention.

[0076] Scheme I describes the synthesis7-(2-methoxyethoxy)-4-piperazinylquinazoline a key intermediate thatwill be utilized in the synthesis of various targets as described inScheme II. The 2-amino-4-fluorobenzoic acid is esterified followed bycyclization with formamide at elevated temperature to afford7-fluoro-4-quinazolinone. The 7-fluoro group is displaced with severalalkoxides generated by treating with NaH in DNM at 100° C. The7-alkoxy-4-quinazolinone is intermediate is converted to7-alkoxy4-chloroquinazoline with thionyl chloride. The key intermediateI was obtained by treating 7-alkoxysubsti-tuted-4-Cl-quinazoline withpiperazine in an appropriate solvent, such as isopropanol, acetonitrile,or THF at room or reflux temperature for 1-6 h in presence of basetriethylamine or pyridine.

[0077] This illustrated Scheme II provides the synthesis of varioussubstituted urea from the intermediate obtained in Scheme I, or by otherprocedures. The reaction of intermediate I with various isocyanatesafforded the final urea compounds. In cases where the isocyanates arenot conunercially available, the piperazine intermediate is treated withphosgene to give carbamoyl chloride intermediate followed by reactionwith various substituted anilines. The piperazine intermediate can alsobe treated with p-nitrophenyl chloroformate to afford nitrophenylcarbamate intermediate that can be treated with various anilines toafford the desired ureas.

[0078] If the urea compound has a terminal NH2 group (or one or more ofthe hydrogen atoms on this amino group is replaced by a displaceablesubstituent), then this compound may be utilized an intermediatecompound with which to produce a urea compound terminated with a—NH-phenyl-R¹ groups. Alternatively, if the a different R¹ group isdesired on the phenyl group, a replaceable para position leaving groupphenyl substituent may be displaced after coupling to provide theparticular R¹ substituent as described for formula I, above.

[0079] Scheme III describes the synthesis of6-(2-methoxyethoxy)-4-piperazinylquinazoline as a key intermediate thatwill be utilized in the synthesis of various positional isomer targets.

[0080] Such procedures for producing the claimed compounds are merely anillustration of a preferred aspect of the invention. Other proceduresand adaptations will be apparent to one of ordinary skill in the artupon views these reaction schemes and the structures of the compoundsaccording to the invention. Such procedures are deemed to be within thescope of the present invention.

[0081] Also, the compounds of formula I and pharmaceutically acceptablesalts thereof may exist in the form of adducts with water (hydrates) orvarious solvents, which are also within the scope of the presentinvention.

[0082] The following non-limiting examples are provided to betterillustrate the present invention.

EXAMPLE 1

[0083] The intermediate 4-piperazinyl-7-(2-piperidylethoxy)quinazolinewas prepared using the procedures as generally described in Scheme 1 asfollows:

[0084] Step A To the ethanol solution (15 mL) of 2-amino-4-fluorobenzoicacid (840 mg, 5.41 mmol) was added thionyl chloride (1.18 mL, 16.23mmol) and the resulting suspension was refluxed overnight. The solventwas evaporated to the residue added EtOAc, washed with 10% NaOHsolution, dried, filtered and evaporated to afford desired ethyl esteras a solid 981 mg, 82%). MS (ES) 184(M+H)

[0085] Step B: To the formamide (6 mL) solution ofethyl-2-amino-4-fluorobenzoate (811 mg, 4.43 mmol) added ammoniumformate (0.450 g, 7.14 mmol) and the reaction mixture was heated at 140°C. overnight. After cooling added water and ethyl acetate. The layerswere separated, the EtOAc layer was dried, filtered and evaporated togive desired quinazolinone (1 g, quantitative). MS (ES) 165 (M+H)

[0086] Step C: To the DMF solution (3 mL) of 1-piperidineethanol (0.689mL, 5.18 mmol) at 0° C. added sodium hydride (0.518 g, 12.95 mmol) andthe mixture was stirred for 30 min. To this cold solution added DMFsolution (3 mL) of intermediate quinazolinone (0.284 g, 1.73 mmol, fromStep B) and the mixture was heated at 75° C. overnight. The solvent wasevaporated and the residue purified by RP-HPLC to afford desired productas a creamy solid (0.459 g, 97%)

[0087] Step D: A mixture of 7-(2-piperidylethoxy)-4-quinazolinone (0.459g, 1.68 mmol), from Step C) and POCl₃ (5 mL) was heated at 75° C.overnight. After cooling excess POCl₃ was removed by evaporation and theresidue azeotroped with toluene to afford intermediate,4-chloro-7-(2-piperidylethoxy)quinazoline (600 mg, 95%)

[0088] Step E: To the isopropanol solution (10 mL) of4-chloroquinazoline (0.615 g, 2.11 mmol, from Step D) added piperazine(0.727 g, 8.44 mmol) and heated the reaction for 4 h at 100° C. Thesolvent was evaporated and the residue purified by RP-HPLC to afford4-piperazinyl-7-(2-piperidylethoxy)quinazoline as a white solid (0.630g,87%).

Example 2

[0089] Preparation of N-(4-cyanophenyl){4-[7-(2-piperidylethoxy)quinazolin-4-yl]piperazinyl}-carboxamide

[0090] To DMF solution (2 mL) of4-piperazinyl-7-(2-piperidylethoxy)quinazoline (from Example 1, Step E,0.287 g, 0.84 mmol) added DMF solution (2mL) of 4-cyanophenylisocyanate(0.181 g, 1.26 mmol) and the reaction was stirred at room temperatureovernight. The solvent was evaporated and residue purified by RP-EHLC toafford desired productN-(4-cyanophenyl){4-[7-(2-piperidylethoxy)quinazolin-4-yl]piperazinyl}carboxamideas a white solid (200 mg, 50%). MS (ES) 487(M+H)

Examples 3-4

[0091] N-(4-cyanophenyl){4-[7-(2-methoxyethoxy)quinazolin-4-yl]piperazinyl}-carboxamide wasprepared using the procedures as described above in Example 1 and 2except that 2-methoxyethanol was used instead of 1-piperidineethanol asan alkoxide anion, to provide the title compound.

[0092] The pharmacological activities of the compounds of the presentinvention are obtained by following the test example procedures asfollows, for example.

Biological Test Assay Type 1

[0093] Inhibitory Effect on Compounds on Autophosphorylation of PlateletDerived Growth Factor β-PDGF Receptor

[0094] (1) HR5 Phosphorylation Assay

[0095] The HR5 cell line is a cell line of CHO cells engineered tooverexpress human β-PDGFR, which cell line is available from the ATCC.The expression level of β-PDGFR in HR5 cells is around 5×10⁴ receptorper cell. For the phosphorylation assay according to the invention, HR5cells were grown to confluency in 96-well microtiter plates understandard tissue culture conditions, followed by serum-starvation for 16hours. Quiescent cells were incubated at 37° C. without or withincreasing concentrations of the test compound (0.01-30 uM) for 30minutes followed by the addition of 8 nM PDGF BB for 10 minutes. Cellswere lysed in 100 mM Tris, pH7.5, 750 mM NaCl, 0.5% Triton X-100, 10 mMsodium pyrophosphate, 50 mM NaF, 10 ug/ml aprotinin, 10 ug/ml leupeptin,1 mM phenylmethylsulfonyl fluoride, 1 mM sodium vanadate, and the lysatewas cleared by centrifugation at 15,000×g for 5 minutes. Clarifiedlysates were transferred into a second microtiter plate in which thewells were previously coated with 500 ng/well of 1B5B11 anti-β-PDGFRmAb, and then incubated for two hours at room temperature. After washingthree times with binding buffer (0.3% gelatin, 25 mM Hepes pH 7.5, 100mM NaCl, 0.01% Tween-20), 250 ng/ml of rabbit polyclonalanti-phosphotyrosine antibody (Transduction Laboratory) was added andplates were incubated at 37° C. for 60 minutes. Subsequently, each wellwas washed three times with binding buffer and incubated with 1 ug/ml ofhorse radish peroxidase-conjugated anti-rabbit antibody (BoehringerMannheim) at 37° C. for 60 minutes. Wells were washed prior to addingABTS (Sigma), and the rate of substrate formation was monitored at 650nm. The assay results are reported as IC₅₀ (expressed as theconcentration of a compound according to the invention that inhibits thePDGF receptor phosphorylation by 50%) as compared to control cells thatare not exposed to a compound according to the invention.

[0096] Examples of such IC₅₀ test results in the HR5 assay for compoundsaccording to the invention are set forth below in Table 1.

[0097] (2) MG63 Phosphorylation Assay

[0098] The MG63 cell line is a human osteosarcoma tumor cell lineavailable from the ATCC. This assay is for measuring endogenous β-PDGFRphosphorylation in MG63 cells. The assay conditions are the same asthose described at for HR5 cell, except that PDGF-BB stimulation isprovided in the presence or absence of 45% human plasma. The HR5 assayresults are reported as an IC₅₀ (expressed as the concentration of acompound according to the invention that inhibits the PDGF receptorphosphorylation by 50%) as compared to control cells that are notexposed to a compound according to the invention.

[0099] Examples of such IC₅₀ test results in the MG63 assay forcompounds according to the invention are set forth below in Table 1.

[0100] The assay results for Compound Examples 1 and 2 are set forth inTable 1 below. TABLE 1 Example MG63 w/human plasma HR5 Compound IC₅₀(μM) IC₅₀ (μM) Example 1 0.103 0.150 Example 2 3.56 2.27

Biological Test Assay Type 2

[0101] Growth Inhibition Against Smooth Muscle cells

[0102] Vascular smooth muscle cells are isolated from a pig aorta byexplanation and used for the test. The cells are put into wells of a96-well plate (8000 cells/well) and cultured in Dulbeccois modifiedEagle's medium (DMEM; Nissui Pharmaceutical Co., Ltd.) containing 10%fetal bovine serum (FBS; Hyclone) for 4 days. Then, the cells arefurther cultured in DMEM containing 0.1% FBS for 3 days, and aresynchronized at the cell growth stationary phase.

[0103] To each well is added DMEM containing 0.1% FBS and a test sampleat a varied concentration, and the cell growth is brought about byPDGF-BB (SIGMA, final concentration: 20 ng/ml). After culturing for 3days, the cell growth is measured using a cell growth assay kit(Boehringer Mannheim) according to the XTT method [J. Immunol. Methods,142, 257-265 (1991)], and the cell growth score is calculated by thefollowing equation.

[0104] Cell growth score=100×{1-(M-PO)/(P100-PO)} whereinP100=absorbance by XTT reagent when stimulated by PDGF-BB; PO=absorbanceby XTT reagent when not stimulated by PDGF-BB, and M=absorbance by XTTreagent after addition of a sample when stimulated by PDGF-BB.

[0105] The test result is expressed as the concentration of a testcompound which inhibits the cell growth by 50% (IC₅₀).

Biological Test Assay Type 3

[0106] Inhibitory Effect on Hypertrophy of Vascular Intima

[0107] Male SD rats (weight: 375-445 g, Charles River, golden standard)are anesthetized with sodium pentobarbital (50 mg/kg, i.p.), and thenthe neck of each animal is incised by the median incision, followed byretrograde insertion of a balloon catheter (2F, Edwards Laboratories)into the left external carotid. After the above treatment is repeatedseven times, the catheter is pulled out, the left external carotid isligated, and the wound is sutured. A test compound is suspended in a0.5% solution of Tween 80 in an aqueous solution of sodium chloride to aconcentration of 20 mg/ml in the case of intraperitoneal administrationand in a 0.5% solution of methyl cellulose 400 to a concentration of 6mg/ml in the case of oral administration. The suspension is administeredonce a day in the case of intraperitoneal administration and once ortwice a day in the case of oral administration for a period of 15 daysstarting on the day before the balloon injury. On the 14th day after theballoon injury, the animal is killed and its left carotid is extirpated.The tissues are fixed with formalin, wrapped in paraffin and sliced,followed by Elastica Wangeeson staining. The area of the cross sectionof the vascular tissues (intima and media) is measured with an imageanalyzer (Luzex F, NIRECO) and the intima/media area ratio (I/M) isregarded as the degree of hypertrophy of the vascular intima.

[0108] From the results obtained, it is apparent when the hypertrophy ofvascular intima is significantly inhibited by administration of thecompounds of the present invention.

Biological Test Assay Type 4

[0109] Evaluation by the Use of a Rat Adjuvant Arthritis Model

[0110] Dead cells of Mycobacterium bacterium (Difco Laboratories Inc.)are disrupted in agate mortar and suspended in liquid paraffin to thefinal concentration of 6.6 mg/ml, followed by sterilization with highpressure steam. Then, 100 ml of the suspension is subcutaneouslyinjected into the right hind foot pad of each animal of groups of female8-weeks-old Lewis rats (Charles River Japan) (6 animals/group) to induceadjuvant arthritis. A test compound is suspended in a 0.5% solution ofmethyl cellulose to the final concentration of 3 mg/ml, and from justbefore the induction of arthritis, the suspension is orally administeredin an amount of 100 ml/100 g of the body weight once a day, 5 days aweek. To a control group is administered a 0.5% solution of methylcellulose. A normal group is given no adjuvant treatment or testcompound administration. The administration of the test compound iscontinued till the 18th day after the adjuvant treatment. On the 17thday, the number of leukocytes in peripheral blood are counted, and onthe 18th day, all the blood is collected, followed by dissection.

[0111] The change in body weight with the passage of time, the change ofedema in hind foot with the passage of time, the weight of spleen andthymus, the number of leukocytes in peripheral blood, the hydroxyprolinecontent of urine, the glucosaminoglycan content of urine, the SHconcentration in serum, the concentration of nitrogen monoxide in serumand the concentration of mucoprotein in serum are measured andevaluated. The volume of each of both hind feet are measured using arat's hind foot edema measurement device (TK-101, Unicom). The number ofleukocytes in peripheral blood are counted using an automaticmultichannel blood cell counter (Sysmex K-2000, Toa Iyo Denshi Co.,Ltd.). The hydroxyproline content of urine is measured according to themethod described in Ikeda, et al., Annual Report of Tokyo MetropolitanResearch Laboratories P. H., 36, 277 (1985), and the glucosaminoglycancontent is measured according to the method described in Moriyama, etal., Hinyo Kiyo, 40, 565 (1994) and Klompmakers, et al., AnalyticalBiochemistry, 153, 80 (1986). The SH concentration in serum is measuredaccording to the method described in Miesel, et al., Iflanmnation, 17,595 (1993), and the concentration of nitrogen monoxide is measuredaccording to the method ofTracey, et al., Journal of Pharmacology &Experimental Therapeutics, 272, 1011 (1995). The concentration ofmucoprotein is measured using Aspro GP Kit (Otsuka Pharmaceutical Co.,Ltd.). The percentage inhibition for each indication is calculatedaccording to the following equation.

% Inhibition={(Control group—Compound-administered group)/(Controlgroup—Normal group)}×100.

[0112] From the results obtain from such assays, it is apparent when thecompound according to the invention inhibits the occurrence of adjuvantarthritis.

Biological Test Assay Type 5

[0113] Activity on a Mesangial Proliferative Glomerulonephritis Model

[0114] Anti-rat Thy-1.1 monoclonal antibody OX-7 (Sedaren) isadministered to male Wister-Kyoto rats (Charles River Japan, 160 g, 6animals/group) in an amount of 1.0 mg/kg by intravenous adnminstrationthrough the tail vein. A test compound is suspended in a 0.5% solutionof methylcellulose and the resulting suspension is administered to eachof the rats twice a day for a period of 7 days starting on the daybefore the administration of OX-7. On the 7th day after the OX-7administration, when mesangial cell growth and extracellular matrixhypeitrophy become prominent, the left kidney of each rat is extirpated,fixed with 20% buffered formalin for 6 hours and wrapped in paraffin,followed by slicing. The obtained pieces are subjected to immune tissuestaining using antibody PC10 (DAKO) against an intranuclear antigen ofproliferative cells. After comparative staining with Methyl Greenstaining solution using diaminobenzidine as a color developer, theparaffin pieces are enclosed. Half of the glomeruli in a kidney pieceare observed and the number of the cells in one glomerulus which arepositive to the intranuclear antigen of proliferative cells arecalculated. The test for the significance of difference is carried outby the Wilcoxon test.

[0115] From such results, it is apparent when the compounds according tothe present invention show alleviating activity on mesangialproliferative glomerulonephritis.

[0116] The compounds of formula (I) and pharmaceutically acceptable-salts thereof can be administered as such, but it is usually preferredto administer them in the form of pharmaceutical compositions, which areused for animals and human beings.

[0117] It is preferred to employ the administration route which is themost effective for the treatment. For example, administration is madeorally or non-orally by intrarectal, intraoral, subcutaneous,intramuscular or intravenous administration.

[0118] Examples of the forms for administration are capsules, tablets,granules, powders, syrups, emulsions, suppositories and injections.

[0119] Liquid compositions such as emulsions and syrups which areappropriate for oral administration can be prepared using water, sugarssuch as sucrose, sorbitol and fructose, glycols such as polyethyleneglycol and propylene glycol, oils such as sesame oil, olive oil andsoybean oil, preservatives such as benzoates, flavors such as strawberryflavor and peppermint, etc.

[0120] Capsules, tablets, powders and granules can be prepared usingexcipients such as lactose, glucose, sucrose and mannitol,disintegrating agents such as starch and sodium alginate, lubricantssuch as magnesium stearate and talc, binders such as polyvinyl alcohol,hydroxypropyl cellulose and gelatin, surfactants such as fatty acidesters, plasticizers such as glycerin, etc.

[0121] Compositions suitable for non-oral administration preferablycomprise a sterilized aqueous preparation containing an active compoundwhich is isotonic to the recipient's blood. For example, injections areprepared using a carrier which comprises a salt solution, a glucosesolution, or a mixture of a salt solution and a glucose solution.

[0122] Compositions for topical application are prepared by dissolvingor suspending an active compound in one or more kinds of solvents suchas mineral oil, petroleum and polyhydric alcohol, or other bases usedfor topical drugs.

[0123] Compositions for intestinal administration are prepared usingordinary carriers such as cacao fat, hydrogenated fat and hydrogenatedfat carboxylic acid, and are provided as suppositories.

[0124] The compositions for non-oral administration may additionally beformulated to contain one or more kinds of additives selected fromglycols, oils, flavors, preservatives (including antioxidants),excipients, disintegrating agents, lubricants, binders, surfactants andplasticizers which are used for the preparation of compositions for oraladministration.

[0125] The effective dose and the administration schedule for each ofthe compounds of formula (I) or a pharmaceutically acceptable saltthereof will vary depending on the administration route, the patient'sage and body weight, and the type or degree of the diseases to betreated. However, it is generally appropriate to administer a compoundof formula (I) or a pharmaceutically acceptable salt thereof in a doseof 0.01-1000 mg/adult/day, preferably 5-500 mg/adult/day, in one toseveral parts.

[0126] All the compounds of the present invention can be immediatelyapplied to the treatment of kinase-dependent diseases of mammals askinase inhibitors, specifically, those relating to tyrosine kinase.Specifically preferred are the compounds which have IC50 within therange of 10 nM-10 μuM. Even more preferred are compounds which have IC50within the range of 10 μM to -1 μM. Most preferred are compounds whichhave an IC50 value which is smaller than 1 μM.

[0127] Specific compounds of the present invention which have anactivity to specifically inhibit one of the three types of proteinkinase (for example, kinase which phosphorylates tyrosine, kinase whichphosphorylates tyrosine and threonine, and kinase which phosphorylatesthreonine) can be selected. Tyrosine kinase-dependent diseases includehyperproliferative malfunction which is caused or maintained by abnormaltyrosine kinase activity. Examples thereof include psoriasis, pulmonaryfibrosis, glomerulonephritis, cancer, atherosclerosis andanti-angiopoiesis (for example, tumor growth and diabetic retinopathy).Current knowledge of the relationship between other classes of kinaseand specific diseases is insufficient. However, compounds havingspecific PTK-inhibiting activity have a useful treatment effect. Otherclasses of kinase have also been recognized in the same manner.Quercetin, genistein and staurosporin, which are all PTK-inhibitors,inhibit many kinds of protein kinase in addition to tyrosine kinase.However, as a result of their lack of the specificity, theircytotoxicity is high. Therefore, a PTK-inhibitor (or an inhibitor ofother classes of kinase) which is apt to bring about undesirable sideeffects because of the lack of selectivity can be identified by the useof an ordinary test to measure cytotoxicity.

[0128] The present invention provides nitrogen-containing heterocycliccompounds and pharmaceutically acceptable salts thereof which inhibitphosphorylation of PDGF receptor to hinder abnormal cell growth and cellwandering and thus are useful for the prevention or treatment ofcell-proliferative diseases such as arteriosclerosis, vascularreobstruction, cancer and glomerulosclerosis.

[0129] Although the present invention has been described in some detailby way of illustration for purposes of clarity of understanding, it willbe apparent to those of ordinary skill in the art that variousmodifications and equivalents can be made without departing from thespirit and scope of the invention. It should be understood that theforegoing discussion and examples merely present a detailed descriptionof certain preferred embodiments. All the patents, journal articles andother documents discussed or cited above are herein incorporated byreference in their entirety.

What is claimed is:
 1. A nitrogen-containing heterocyclic compound ofthe formula:

wherein R¹ is a member selected from the group consisting of: —CN,—O—C₁₋₈ alkyl that is a straight or branched chain, —O-phenyl,—O-naphthyl, —O-indolyl, and —O-isoquinolinyl; R² and R⁴ are eachindependently a member selected from the group consisting of: hydrogen,—O(—CH₂)—CH₃, —O—CH₂—CH═CH₂, —O—CH₂—C4CH and —O(—CH₂)_(n)—R³; providingthat one of the R² and R⁴ groups is hydrogen and the remaining R² or R⁴group is other than hydrogen; n is 1,2or3; R₃ is a member selected fromthe group consisting of: —OH, —O—CH₃, —O—CH₂—CH₃, —NH₂, —N(—CH₃)₂,—NH(—CH₂-phenyl),—NH(-Phenyl),—CN

and all pharmaceutically acceptable isomers, salts, hydrates, solvatesand prodrug derivatives thereof.
 2. The compound of claim 1, wherein R¹is a member selected from the group consisting of CN, —O-methyl,—O-ethyl, —O-propyl, —O-isopropyl, —O-butyl, —O-t-butyl, —O-isoamyl,1-naphthyloxy, 2-naphthyloxy, 4-indolyloxy, 5-indolyloxy,5-isoquinolyloxy, and position isomers and homologs thereof, and allpharmaceutically acceptable isomers, salts, hydrates, solvates andprodrug derivatives of such compounds.
 3. The compound of claim 1,having formula I(a) or formula I(b) as follows:

wherein R¹ is a member selected from the group consisting of: —CN, —O—C,alkyl that is a straight or branched chain, —O-phenyl, —O-naphthyl,—O-indolyl and —O-isoquinolinyl; and all pharmaceutically acceptableisomers, salts, hydrates, solvates and prodrug derivatives thereof. 4.The compound of claim 3, wherein R¹ is a member selected from the groupconsisting of CN, —O-methyl, —O-ethyl, —O-propyl, —O-isopropyl,—O-butyl, —O-t-butyl, —O-isoamyl, 1-naphthyloxy, 2-naphthyloxy,4-indolyloxy, 5-indolyloxy, 5-isoquinolyloxy, and position isomers andhomologs thereof, and all pharmaceutically acceptable isomers, salts,hydrates, solvates and prodrug derivatives of such compounds.
 5. Thecompound of claim 1, having formula I(c) or formula I(d) as follows:

wherein R¹ is a member selected from the group consisting of: —CN,—O—C₁₋₈ alkyl that is a straight or branched chain, —O-phenyl,—O-naphthyl, —O-indolyl and —O-isoquinolinyl; and all pharmaceuticallyacceptable isomers, salts, hydrates, solvates and prodrug derivativesthereof.
 6. The compound of claim 5, wherein R¹ is a member selectedfrom the group consisting of CN, —O-methyl, —O-ethyl, —O-propyl,—O-isopropyl, —O-butyl, —O-t-butyl, —O-isoamyl, 1-naphthyloxy,2-naphthyloxy, 4-indolyloxy, 5-indolyloxy, 5-isoquinolyloxy, andposition isomers and homologs thereof, and all pharmaceuticallyacceptable isomers, salts, hydrates, solvates and prodrug derivatives ofsuch compounds.
 7. The compound of claim 1, having formula I(e) orformula I(f) as follows:

wherein R¹ is a member selected from the group consisting of: —CN,—O—C₁₋₈ alkyl that is a straight or branched chain, —O-phenyl,—O-naphthyl, —O-indolyl and —O-isoquinoliniyl; and all pharmaceuticallyacceptable isomers, salts, hydrates, solvates and prodrug derivativesthereof.
 8. The compound of claim 7, wherein R¹ is a member selectedfrom the group consisting of CN, —O-methyl, —O-ethyl, —O-propyl,—O-isopropyl, —O-butyl, —O-t-butyl, —O-isoamyl, 1-naphthyloxy,2-naphthyloxy, 4-indolyloxy, 5-indolyloxy, 5-isoquinolyloxy, andposition isomers and homologs thereof, and all pharmaceuticallyacceptable isomers, salts, hydrates, solvates and prodrug derivatives ofsuch compounds.
 9. The compound of claim 1, having formula I(g) orformula I(h) as follows:

wherein n is 1, 2 or 3; R¹ is a member selected from the groupconsisting of: —CN, —O—C₁₋₈ alkyl that is a straight or branched chain,—O-phenyl, —O-naphthyl, —O-indolyl and —O-isoquinolinyl; R³ is a memberselected from the group consisting of: —OH, —O—CH₃, —O—CH₂—CH₃, —NH2,—N(—CH₃)₂, —NH(—CH₂-phenyl), —NH(-Phenyl),—CN

and all pharmaceutically acceptable isomers, salts, hydrates, solvatesand prodrug derivatives thereof.
 10. The compound of claim 9, wherein R¹is a member selected from the group consisting of CN, —O-methyl,—O-ethyl, —O-propyl, —O-isopropyl, —O-butyl, —O-t-butyl, —O-isoamyl,1-naphthyloxy, 2-naphthyloxy, 4-indolyloxy, 5-indolyloxy,5-isoquinolyloxy, and position isomers and homologs thereof, and allpharmaceutically acceptable isomers, salts, hydrates, solvates andprodrug derivatives of such compounds.
 11. A compound according to claim1, which is a compound selected from the group consisting of:N-[4-(methylethoxy)phenyl]{4-[7-(2-morpholin-4-ylethoxy)quinazolin-4-yl]piperazinyl}carboxamide

N-(4-cyanophenyl){4-[7-(2-morpholin-4-ylethoxy)quinazolin-4-yl]piperazinyl}carboxamide

N-(4-cyanophenyl){4-[7-(2-pyrrolidinylethoxy)quinazolin-4-yl]piperazinyl}carboxamide

N-[4-(methylethoxy)phenyl]{4-[7-(2-pyrrolid inylethoxy)quinazolin-4-yl]piperazinyl}carboxamide

N-[4-(methylethoxy)phenyl]{4-[7-(2-piperidylethoxy)quinazolin-4-yl]piperazinyl}carboxamide

{4-[7-(2-methoxyethoxy)quinazolin-4-yl]piperazinyl}-N-[4-(methylethoxy)phenyl]carboxamide

N-(4-cyanophenyl){4-[7-(2-methoxyethoxy)quinazolin-4-yl]piperazinyl}carboxamide

N-(4-cyanophenyl){4-[7-(2-piperidylethoxy)quinazolin-4-yl]piperazinyl}carboxamide

{4-[7-(2-methoxyethoxy)quinazolin-4-yl]piperazinyl}-N-(4-phenoxyphenyl)carboxamide

N-(4-indol-5-yloxyphenyl){4-[7-(2-methoxyethoxy)quinazolin-4-yl]piperazinyl}carboxamide

N-(4-(5-isoquinolyloxy)phenyl){4-[7-(2-methoxyethoxy)quinazolin-4-yl]piperazinyl}carboxamide

N-[4-(methylethoxy)phenyl]{4-[7-(3-piperidylpropoxy)quinazolin-4-yl]piperazinyl}carboxamide

{4-[7-(2-methoxypropoxy)quinazolin-4-yl]piperazinyl}-N-(4-phenoxyphenyl)carboxamide

N-(4-indol-5-yloxyphenyl){4-[7-(2-methoxypropoxy)quinazolin-4-yl]piperazinyl}carboxamide

N-(4-(5-isoquinolyloxy)phenyl){4-[7-(2-methoxypropoxy)quinazolin-4-yl]piperazinyl}carboxamide

N-(4-cyanophenyl){4-[7-(3-piperidylpropoxy)quinazolin-4-yl]piperazinyl}carboxamide

N-[4-(methylethoxy)phenyl]{4-[7-(3-morpholin-4-ylpropoxy)quinazolin-4-yl]piperazinyl}carboxamide

N-(4-cyanophenyl){4-[7-(3-morpholin-4-ylpropoxy)quinazolin-4-yl]piperazinyl}carboxamide

N-[4-(methylethoxy)phenyl]{4-[7-(3-pyrrolidinylpropoxy)quinazolin-4-yl]piperazinyl}carboxamide

N-(4-cyanophenyl){4-[7-(3-pyrrolidinyipropoxy)quinazolin-4-yl]piperazinyl}carboxamide

N-(4-cyanophenyl){4-[7-(3-methoxypropoxy)quinazolin-4-yl]piperazinyl}carboxamide

{4-[7-(3-methoxypropoxy)quinazolin-4-yl]piperazinyl}-N-[4-(methylethoxy)phenyl]carboxamide

and all pharmaceutically acceptable isomers, salts, hydrates, solvatesand prodrug derivatives thereof.
 12. A pharmaceutical compositioncomprising an effective amount of a nitrogen-containing heterocycliccompound according to claim 1, or a pharmaceutically acceptable saltthereof, and a pharmaceutically acceptable diluent or carrier.
 13. Amethod of inhibiting phosphorylation of PDGF receptor in a patientcomprising administering a composition according to claim 12 to thepatient.
 14. A method for inhibiting abnormal cell growth and cellwandering in a patient and thereby preventing or treating acell-proliferative disease, comprising the step of administering acomposition according to claim 12 to the patient.
 15. A method accordingto claim 14, wherein said cell-proliferative disease is selected fromthe group consisting of arteriosclerosis, vascular re-obstruction,restenosis, cancer and glomerulosclerosis.